Methods For Detection Of MiRNAs And Oxidase Substrates Based On Homogeneous Enzymatic Chemiluminescence

Chemiluminescence has many advantages including high sensitivity,simple equipments,fast analysis,wide linear range,easy auto-operation and so on.From 1980 s,chemiluminescence has been widely researched and applied in various fields,such as chemical analysis,biological analysis,environmental analysis and medical diagnosis.For the past few years,the selectivity,the sensitivity and application of chemiluminometry combined with new technologies?biological enzyme,nucleic acid,nanomaterials and so on?has been sharply increased.Two methods for detection of mi RNA and oxidase substrates in the serum based on luminol-H2O2 system catalyzed by G-quadruplexes DNAzymes and horseradish peroxidaes?HRP?have been developed.The thesis concerned two research sections:1.A new method for low backgrounding sensing miRNA based on both-end blocked G-quadruplexes DNAzymes was builded.The both ends of probe DNA were pre-blocked by a block DNA which can effectively prohibit the probe from forming intramolecular or intermolecular G-quadruplexes DNAzymes,and the inactived G-quadruplexes DNAzymes can easily actived by targets though strand displacement reaction between block DNA and target mi RNA after target miRNA was added into the system.This method had high blocking and activation efficiencies?97% and 92.8 respectively?and attained a higher ratio of signal to noise compared with other general methods which were reported by literatures without any amplifications or label steps.The block and actived processes were verified and explained by labeled DNA,ultraviolet absorption spectrum,chemiluminescence,fluorescence spectrum and other analytical methods.The chemiluminescence intensity was linearly dependent on the amount of mi RNA-21 in the range of 25 pM to 1 nM with a detection limit of approximately 8 pM based on 3? after optimising the block II length of block DNA,the concentrations of probe DNA,block DNA,NH₄⁺,k⁺,Hemin and both-end blocked beacon.To investigate the applicability of this sensing system,spiked human serums and HEPES buffer with the same concentrations of miRNA-21 were detected by chemiluminescent detector after mi RNA was extracted by miRNA miniprep Kit.The results demonstrated that the serum matrix did not yield a significant influence for miRNA detection.The proposed method also can accurately detect the concentrations of miRNA let-7a and target DNA by changing the recognition region sequences of probe DNA.The system was promising for detection of small biological molecules and proteins if the recognition region sequences were replaced by appropriate aptamers.2.A bienzyme-quantum dots?QDs?chemiluminescence resonance energy transfer?CRET?system which was developed by coupling both oxidase?glucose oxidase and cholesterol oxidase?and horseradish peroxidase?HRP?onto QDs which was synthesized by hydrothermal method to detect multiple oxidase substrates.The bienzyme allowed fast in situ cascaded H2O2 generation and consumption,thus alleviating fluorescence quenching of QDs.The nanosized QDs accommodate the two enzymes in a nanometric range,and the CL reaction was confined on the surface of QDs accordingly,thereby amplifying the CL reaction rate and improving CRET efficiency.As a result,CRET efficiency of 39.7% was obtained;the highest CRET efficiency by far was obtained using QDs as the energy acceptor.Under the optimum conditions,the proposed CRET system could be explored for ultrasensitive sensing of various oxidase substrates?here exemplified with cholesterol and glucose?.A linear range from 10 nM and 1000 n M was obtained with a detection limit of 3.4 nM for glucose.A linear relationship was found in the range of 2.5 nM to 500 nM for free cholesterol.Limits of detection?LOD,3??for free cholesterol and glucose were found to be 0.8 and 3.4,respectively.At last,we detected the concentrations of glucose and free cholesterol in the human serums.The measured valves were in good agreement with the reference valves and with spiked recoveries ranging from 96 to 116%.It demonstrated that the bienzyme-QDs system can give accurate concentrations of glucose and free cholesterol in the human serums and was promising for potential clinical diagnosis of hyperglycemia and hyperlipemia.